• Toxicological Research
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• 제16권4호
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• pp.269-274
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• 2000

Epoxidised soya bean oil (ESBO, 1000, 2000 or 4000 mg/kg) was orally administered to BALB/c mice daily for 28 consecutive days, and the control mice were exposed to vehicle (corn oil). Mice were immunized and challenged with sheep red blood cells (SRBC) or bovine serum albumin (BSA). In groups exposed to ESBO, the body weight gains and the relative lymphoid organ weights were not significantly changed as compared with control group. Secondary IgG antibody response to BSA was not significantly changed by ESBO, but plaque-forming cell (PFC) response to SRBC was significantly suppressed in mice treated with 4000 mg ESBO/kg/day. The mitogenic response of splenic B cells induced by LPS was not effected by ESBO in any of the groups. These results indicate that ESBO did not induce significant humoral immune response at a dose less than 2000 mg/kg/day in mice.

• 한국수정란이식학회지
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• 제15권3호
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• pp.271-277
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• 2000

This study was conducted to establish the optimal culture conditions for in vitro production of bovine embryos derived from slaughter house ovaries. Cumulus-oocyte- complexes (COCs) collected by aspiration from follicles of 2~7 mm in diameter were matured in Ham's F-10 medium supplemented with 0.01 $\mu\textrm{g}$/m1 epidermal growth factor (EGF) at 39$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. After 24 hrs of culture, the oocytes were co-cultured with epididymal sperm selected off by Percoll-density gradient in TALP medium for 24 hrs. The presumptive zygotes were cultured in HECM-6 medium for 3 d post-insemination, and followed by cultured in TCM199 medium until 7 to 10d post-insemination. The cultures were compared of their cleavage and development into later stage in culture medium by additions of different protein sources (PVA, BSA and BCS) and by different embryo density. The rates of cleavage and development rates into blastocyst were not significantly (P<0.05) different among the culture media containing with BSA (75.0% and 40.5%), BCS (76.7% and 38.0%) and PVA (72.5% and 42.2%), respectively. Significantly (P<0.05) higher blastocysts rates were obtained in culturing of 30 and 40 embryos in each 50$\mu$l droplets of culture medium than in 5, 10 and 20 embryos. These results indicate that the optimal density of embryos is 30~40 embryos in a 50$\mu$l droplet of culture medium. Furthermore there is no effect of different protein sources on early embryonic development.

• Journal of Pharmaceutical Investigation
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• 제41권2호
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• pp.95-102
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• 2011

An amphiphilic cationic branched methoxy poly (ethylene glycol)-branched polyethylenimine - poly(L-phenylalanine) (mPEG-bPEI-pPhe) block copolymer was successfully synthesized by ring-opening polymerization (ROP) of N-carboxyanhydride of L-phenylalanine (Phe-NCA) with mPEG-bPEI for the preparation of more stable polyelectrolyte complex (PEC) included a hydrophobic interaction. mPEG-bPEI was firstly prepared by the coupling of mPEG and bPEI using hexamethylene diisocyanate (HMDI). The structural properties of mPEG-bPEI-pPhe copolymers were confirmed by $^1H$ NMR. The copolymers exhibited a self-assemble behavior in water above critical aggregate concentration (CAC) in the range of 0.01-0.14 g/L. The CAC of copolymers obviously depended on the hydrophobic block content in the copolymers (the value decreased with the increase of the pPhe block content). The cationic copolymers have the ability to form multi-interaction complex (MIC) with bovine serum albumin (BSA) and plasmid DNA through multi-interaction (electrostatic and hydrophobic interaction). The physicochemical characterization of the complex was carried out by the measurement of zeta potential and particle size. Their zeta-potentials were positive (approximately +10 mV) and their sizes decreased with increasing pPhe contents in the copolymers (PPF/BSA wt% ratio = 2). The complex showed good stability at high ionic strength. Therefore, mPEG-bPEI-pPhe block copolymer was considered as a potential material to enhance the stability of complex including biotherapuetic drugs.

• Journal of Microbiology
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• 제35권2호
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• pp.109-116
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• 1997

An extracellular proteinase of Candida albicans was purified by a combination of 0~75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephacryl S-200 HR molecular sieve chromatography. Its mlecular weight was approximately 41 kDa on SDS-PAGE and isoelectric point was 4.4. The enzyme was inhibited by pepstain A. Optimum enzyme activity ranged from pH 2.0 to 3.5 with its maximum at pH 2.5 and a temperature of 45$^{\circ}C$. The addition of divalent cations, $Ca^{2+}$, Zn$^{2+}$ and $Mg^{2+}$, resulted in no significant inhibition of enzymatic activity. However, some inhibitory effects were observed by Fe$^{2+}$, Ag$^{2+}$ and Cu$^{2+}$. With BSA as substrate, an apparent $K_m$ was determined to be 7$\times$10$^{-7}$ M and $K_i$, using pepstatin A as an inhibitor, was 8.05$\times$10$^{-8}$ M. N-terminal amino acid sequence was QAVPVTLXNEQ. Degradation of BSA and fibronectin was shown but not collagen, hemoglobin, immunoglobulin G, or lysozyme. The enzyme preferred peptides with Glu and Leu at the P$_1$ position, but the enzyme activity was highly reduced when the P$_2$ position was phe or pro. This enzyme showed antigenicity against sera of patients with candidiasis.

• Toxicological Research
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• 제27권2호
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• pp.125-131
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• 2011

Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1-carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with ${\lambda}$-type light chains. The $IC_{50}s$ of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml ($R^2$ > 0.99) and from 1 to 100 ng/ml ($R^2$ > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed.

• 대한미생물학회지
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• 제22권4호
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• pp.377-392
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• 1987

Monoclonal antibody to the Escherichia coli heat-stable enterotoxin(ST) was produced to develop a rapid and convenient diagnostic method to the ST. The toxin was purified from culture supernatant of enterotoxigenic E. coli O148H28($ST^+/LT^+$) and conjugated to bovine serum albumin(BSA). The ST-BSA conjugate was used to immunize BALB/c mice and the immune spleen cells from these mice were fused with $P3{\times}63$ Ag8.V653 plasmacytoma cells. Hybridomas were screened by ELISA and positive hybridomas were cloned by limiting dilution. Finally, one stable clone (AS36) specific to ST was selected for further growth and characterization. Antibody titers of culture supernatant and ascitic fluid from BALB/c mice were 1:1,024 and 1:20,480 respectively in ELISA. The isotype and subclass of monoclonal antibody was IgG1 in sandwich ELISA. To test the neutralizing effect of monoclonal antibody on toxin activity of ST, mixture of ascitic fluid and ST was assayed by infant mouse assay and this monoclonel antibody was proved to be a neutralizing antibody. The titer of ascitic fluid which completely neutralized biological activity of 4 units of ST was 1:4. Purified ST was quantitatively measured by competitive ELISA and minimum amount of ST detectable by this assay was 250pg, which was an amount six-fold smaller than that detectable by infant mouse assay. Four reference strains of enterotoxigenic E. coli from WHO were detected by competitive ELISA and highly specific, sensitive and reproducible result was obtained.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.67-67
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• 2003

Early development of porcine oocytes fertilized in vitro was examined in different culture conditions. Porcine ovaries were collected from local slaughter-house. Cumulus-oocytes complexes were aspirated from 2 to 6 mm follicles. The collected oocytes were cultured for in vitro maturation in NCSU-23 medium with 5 mM hypotaurine, 0.57 mM cystein, 10% porcine follicle fluid, 10 IU/ml PMSG and 10 IU/ml hCG for 42~44 hrs. The frozen-thawed spermatozoa were washed by centrifigation 2 times at 1, 500 rpm in D-PBS with 5.56 mM glucose, 0.33 mM Na-pyruvate, 100 IU/ml penicillin, 1$\mu\textrm{g}$/ml streptomycin and 1ng/ml BSA. The fertilization medium used mTBM with 2 mM caffeine and 2 mg/ml BSA and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to 2.5$\times$10$^{6}$ cells/ml motile sperm during fertilization in vitro. At 8hrs h after insemination, the oocytes were transferred into NCSU-23 medium with 5.0 mM hypotaurine and 4 mg/ml BSA and cultured for 7 days. In first experiment, the mean numbers of oocytes collected from 20 ovaries were 674.4 oocytes, and 4.1(27.6), 12.5(84.0), 25.4(171.6) and 57.9%(390.8) for A, B, C and D grade in morphological classification. In the second experiment, when culture medium was supplemented with various concentrations of EGF, the proportions of oocytes cleaved were 56.9, 55.7, 61.9 and 54.7% for 0, 5, 10 and 20ng/ml EGF. The higher proportions(15.1%) of oocytes developed to morular stage were obtained at concentration of 10ng/ml than 0 and 5ng/ml EGF (P<0.05). However, the proportions of embryos developed to blastocyst stage were not significantly different among concentrations of EGF. In another experiment, when the medium supplemented with catalase was used, the proportions of oocytes cleaved were higher in the concentration of 0 unit (56.5%, 61/108) than 100 and 1, 000 unit/ml of catalase (P<0.05). Although the developmental capacity of embryos was improved by medium with 0 unit/ml compared with 100, 500 and 1, 000 units/ml of catalase in oocytes developed to morula and blastocyst stages, were not significantly different among concentrations of catalase.

• 농약과학회지
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• 제5권2호
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• pp.1-12
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• 2001

다클론항체를 이용하여 환경 시료중 유기인계 살충제 acephate를 분석할 수 있는 경합적 간접효소면역측정법(competitive indirect ELISA)을 개발하기 위하여 분석물과 화학구조가 유사하며 hexanoic acid를 지닌 3종의 hapten을 합성하였다. 이들 hapten을 active ester 방법으로 담체단백질인 keyhole limpet hemocyanin과 접합시켜 면역원으로 사용하있고, bovine serum albumin과 접합시켜 homologous 혹은 heterologous ELISA를 위한 코팅항원으로 사용하였다. 생산된 항체들과 코팅항원을 최종선발하여 acephate 잔류분석을 위한 ELISA를 위하여 최적화하였다. 단백질의 농도가 $1{\mu}g/mL$인 코팅항원 (hapten-3-BSA)과 다클론항체 #8377를 16000배로 희석한 heterologous ELISA에서 최적화된 acephate의 $IC_{50}$ 값은 110 ng/mL이었고, 분석범위와 최소검출한계는 각각 $10{\sim}1000$과 4 ng/mL 이었다. Acephate의 주분해산물이고 살충제로 사용되는 methamidophos을 위시한 몇몇 유기인계 살충제의 항체에 대한 교차반응은 모두 0.02%이하였다. 이와 같은 결과는 본 ELISA가 농산물 및 환경시료중의 acephate 잔류분석을 위하여 편리한 방법이 될 수 있음을 시사한다.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.80-80
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• 2002

This study was carried out that to investigate the effects of amino acids supplemented with culture medium on development of porcine embryos cultured in vitro. Cumulus oocyte complexes (COCs) were cultured in the maturation medium containing hormones (0.5$\mu\textrm{g}$/$m\ell$ LH, 0.5$\mu\textrm{g}$/$m\ell$ FSH and 1$\mu\textrm{g}$/$m\ell$ estradiol-17${\beta}$) for 20-22 h at 39$^{\circ}C$ in an atmosphere of 5% CO$_2$in air. Subsequently, COCs were cultured in hormone-free maturation medium for 20-22 h. After maturation for 40-44h, oocytes were removed cumulus cells by pipetting and cultured with epididymal sperm for 5 h in the mTBM. Embryos obtained were divided in 4 groups (1) cultured in NCSU 23 containing 0.4% BSA to blastocyst stage(Control), (2) essential amino acids (EA), (3) non-essential amino acids (NA), (4) mixture of essential and non essential amino acid (EA+NA). All treated groups(2-4) were used a glucose free NCSU 23 medium supplemented with pyruvate (0.33 mM), lactate (4.5 mM) to morula stage. From morula to blastocyst stage embryos of all treated groups were cultured in NCSU 23 containing 0.4% BSA. The rates of cleaved oocytes at 48 h after IVF were from 82% to 88% in the groups of control, EA, NA and EA+NA, respectively. The in vitro developmental rates into blastocysts in the groups of EA and EA+NA were significantly (P<0.05) higher than those of group of control (35.1, 35.4 vs. 19.4%, respectively), however, no significant (P<0.05) between control and NA. In conclusion, supplemented with essential amino acid or mixture of essential and non essential amino acid in the culture medium at morula stage increased the rate of development to blastocyst on in vitro produced porcine embryos.

• 한국어병학회지
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• 제24권2호
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• pp.153-160
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• 2011

본 구에서는 백신을 넙치 양식 현장에 적용하기 위한 기초연구로서 항원의 투여방법 및 항원 투여 후 사육환경 변화에 따른 넙치의 특이 항체 반응에 대한 영향을 조사하였다. 항원의 투여방법에 따른 항체 반응을 조사하기 위해 넙치에 BSA를 복강 및 근육 주사한 후 주기적으로 혈액을 취해 BSA에 대한 항체기를 측정한 결과, 근육 주사법이 복강 주사법보다 항체형성 초기시점에서 약간 높은 항제기를 나타났으나 그 이후의 항체 반응에는 유의적인 차이가 관찰되지 않아, 백신을 투여하는 방법으로 근육주사법 뿐만 아니라 복강 주사법도 유용하게 사용될 수 있을 것으로 사료되었다. 사육환경 변화에 따른 백신의 영향을 조사하기 위해 BSA를 면역시킨 넙치에 사육수온의 급하강, handling 및 중유에 노출시킨 후 항체기를 측정한 결과, 수온변화 및 handling 실험구가 양성 대조구보다 항체형성 초기시점에서 약간 높은 항체기를 나타났으나, 그 이후의 항체 반응에는 서로간의 유의적인 치아는 관찰되지 않아, 위의 환경 변화는 넙치의 특이 항체반응에 영향을 거의 끼치지 않는 것으로 확인되었다. 이상의 결과, 넙치 양식현장에서 백신은 일시적인 수온변화, 중유노출 및 handling에 의한 환경변화에서도 유용하게 사용 될 수 있을 것으로 사료되었다.